Anti-EGFR antibody and anti-c-Met/anti-EGFR bispecific antibodies comprising the same

ABSTRACT

An anti-EGFR scFV fragment, an anti-c-Met/anti-EGFR bispecific antibody including the same, and a method of preventing and/or treating a cancer using the same are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No.10-2013-0089280 filed on Jul. 29, 2013 in the Korean IntellectualProperty Office, the entire disclosure of which is hereby incorporatedby reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: 144,062 bytes ASCII (Text) file named“718050_ST25_revised_20151120” created Nov. 20, 2015.

BACKGROUND

1. Field

Provided are an anti-EGFR antibody or antigen binding fragment thereof,an anti-c-Met/anti-EGFR bispecific antibody including the same, and amethod of preventing and/or treating a cancer using the same.

2. Description of the Related Art

c-Met and EGFR (or HER family) interact with each other and are involvedin various mechanisms related to tumors. These proteins (targets) aretypical receptor tyrosine kinases (RTKs) present at the surface ofcells, thereby inducing the proliferation of cancer cells, thepenetration of the cancer cells, angiogenesis, etc. Also, these proteinsparticipate in each other's signal transduction systems by interactingwith each other, thereby inducing resistance against each other'stherapeutic agents.

Meanwhile, multispecific antibodies targeting two or more antigens havebeen developed in various kinds and forms and are expected as a new drugantibody having excellent therapeutic effects compared to a monoclonalantibody. Most multispecific antibodies have been developed so thattheir therapeutic effects on cancers can be increased by recognizing anantigen of cytotoxic cells (killer cells) and another antigen of cancercells at the same time thus allowing the cancer cells to be killed bythe cytotoxic cells. However, when considering that the research resultsreveal that cancer cells themselves can be mutated to proliferate andpenetrate even by intracellular ligands or various antigens of the samecancer cells other than the targeted antigen, it is expected that amultispecific antibody capable of recognizing another antigen of thecancer cells as well as an antigen of the killer cells will be alsouseful in treating cancers.

Accordingly, there is a need for the development of a multispecificantibody which is predicted to achieve effective cancer treatment byrecognizing two or more kinds of antigens in cancer cells at the sametime (e.g., a bispecific antibody).

SUMMARY

One embodiment provides a polypeptide including one amino acid sequenceor a combination of two or more amino acid sequences selected from thegroup consisting of SEQ ID NO: 109 to SEQ ID NO: 114.

Another embodiment provides an anti-EGFR antibody or an antigen-bindingfragment thereof comprising or consisting essentially of at least oneheavy chain complementarity determining region selected from the groupconsisting of CDR-H1 including the amino acid sequence of SEQ ID NO:109, CDR-H2 including the amino acid sequence of SEQ ID NO: 110, andCDR-H3 including the amino acid sequence of SEQ ID NO: 111; at least onelight chain complementarity determining region selected from the groupconsisting of CDR-L1 including the amino acid sequence of SEQ ID NO:112, CDR-L2 including the amino acid sequence of SEQ ID NO: 113, andCDR-L3 including the amino acid sequence of SEQ ID NO: 114; or acombination of the at least one heavy chain complementarity determiningregion and the at least one light chain complementarity determiningregion.

Another embodiment provides an anti-c-Met/anti-EGFR bispecific antibodyincluding an anti-c-Met antibody or an antigen-binding fragment thereofand an anti-EGFR antibody or an antigen-binding fragment thereof,wherein the anti-c-Met antibody or an antigen-binding fragment thereofspecifically binds to an epitope including 5 or more contiguous aminoacids within SEMA domain (SEQ ID NO: 79) of c-Met protein, and theanti-EGFR antibody or an antigen-binding fragment thereof includes atleast one heavy chain complementarity determining region selected fromthe group consisting of CDR-H1 including the amino acid sequence of SEQID NO: 109, CDR-H2 including the amino acid sequence of SEQ ID NO: 110,and CDR-H3 including the amino acid sequence of SEQ ID NO: 111; at leastone light chain complementarity determining region selected from thegroup consisting of CDR-L1 including the amino acid sequence of SEQ IDNO: 112, CDR-L2 including the amino acid sequence of SEQ ID NO: 113, andCDR-L3 including the amino acid sequence of SEQ ID NO: 114; or acombination of the at least one heavy chain complementarity determiningregion and the at least one light chain complementarity determiningregion.

Another embodiment provides a pharmaceutical composition including thepolypeptide or the anti-c-Met/anti-EGFR bispecific antibody as an activeingredient.

Another embodiment provides a pharmaceutical composition for preventingand/or treating a cancer including the anti-c-Met/anti-EGFR bispecificantibody as an active ingredient.

Another embodiment provides a method of preventing and/or treating acancer including administering a pharmaceutically effective amount ofthe anti-c-Met/anti-EGFR bispecific antibody to a subject in need ofpreventing and/or treating a cancer.

Additional compositions and methods are described in the followingsections.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic of an anti-c-Met/anti-EGFR bispecific antibodyaccording to an embodiment.

FIG. 2 is a size exclusion chromatography displaying a monomericproperty of the purified anti-c-Met/anti-EGFR bispecific antibodyaccording to an embodiment.

FIG. 3 is a graph displaying the binding affinities to EGFR and c-Met ofan anti-c-Met/anti-EGFR bispecific antibody according to an embodiment.

FIG. 4A is a graph displaying the cell growth of various cancer cellstreated with antibodies including an anti-c-Met/anti-EGFR bispecificantibody according to an embodiment, which is calculated as a relativevalue to that of a control (media, without antibody).

FIG. 4B is a graph displaying the cell growth of various cancer cellstreated with antibodies including an anti-c-Met/anti-EGFR bispecificantibody according to an embodiment, which is calculated as a relativevalue to that of a control (media, without antibody).

FIG. 4C is a graph displaying the cell growth of various cancer cellstreated with antibodies including an anti-c-Met/anti-EGFR bispecificantibody according to an embodiment, which is calculated as a relativevalue to that of a control (media, without antibody).

FIG. 4D is a graph displaying the cell growth of various cancer cellstreated with antibodies including an anti-c-Met/anti-EGFR bispecificantibody according to an embodiment, which is calculated as a relativevalue to that of a control (media, without antibody).

FIG. 4E is a graph displaying the cell growth of various cancer cellstreated with antibodies including an anti-c-Met/anti-EGFR bispecificantibody according to an embodiment, which is calculated as a relativevalue to that of a control (media, without antibody).

FIG. 4F is a graph displaying the cell growth of various cancer cellstreated with antibodies including an anti-c-Met/anti-EGFR bispecificantibody according to an embodiment, which is calculated as a relativevalue to that of a control (media, without antibody).

FIG. 5 is a graph displaying the cell growth of resistance-induced cells(HGF treated N87 cell (upper) and NCI-H820 cell (lower)) when treatedwith antibodies including an anti-c-Met/anti-EGFR bispecific antibodyaccording to an embodiment.

FIG. 6 is an immunoblot showing the phosphorylation and degradation ofc-Met and EGFR when treated with antibodies including ananti-c-Met/anti-EGFR bispecific antibody according to an embodiment.

FIG. 7 provides fluorescence microscopic images of SNU638 gastric cancercells showing co-localization of c-Met and EGFR after treating ananti-c-Met/anti-EGFR bispecific antibody according to an embodiment.

FIG. 8 consists of graphs displaying the expression levels of c-Met andEGFR when treated with an anti-c-Met antibody or withanti-c-Met/anti-EGFR bispecific antibody according to an embodiment,indicating the degradation of both of c-Met and EGFR by theanti-c-Met/anti-EGFR bispecific antibody.

FIG. 9 consists of graphs displaying comparative cell migration level ofSNU638 gastric cancer cells when treated with an anti-c-Met/anti-EGFRbispecific antibody according to an embodiment which is calculated as arelative value to that of a control (media, without antibody).

FIG. 10 is a graph displaying tumor volume change in a mouse xenograftmodel when treated with an anti-c-Met/anti-EGFR bispecific antibodyaccording to an embodiment showing in vivo anti-tumor efficacy ofbispecific antibody in animal cancer model.

DETAILED DESCRIPTION

The existing drugs which recognize only EGFR, a typical target widelyexpressed on cancer cells, induce over-expression and mutation of c-Met,allowing cancer cells to acquire resistance against the drugs, wherebythe therapeutic effects of the drugs could be reduced. In this regard,in the present invention, it is verified that a bispecific antibodyrecognizing c-Met and EGFR at the same time prevents the development ofresistance and shows excellent cancer cell inhibitory effects, even incancer cells having resistance, by previously blocking c-Met-implicatedsignal transduction which causes resistance against drugs.

Although various bispecific antibodies have been developed, theirefficiency was not proved in clinical tests or their several sideeffects were observed. For these reasons, there were many cases whichwere not approved by FDA and were not marketed as therapeuticantibodies. In spite of the fact that bispecific antibodies havingvarious forms and mechanisms have been developed, the bispecificantibodies were not marketed due to a problem in the stability andproductivity of the antibodies. In the production of early bispecificantibodies having an IgG form, due to random combination between lightchains and heavy chains of antibodies, it is very difficult to separateand purify a desired kind of bispecific antibody, which becomes anobstacle in the mass production. Also, in the case of bispecificantibodies with non-IgG forms, their stabilities as a drug were notverified in protein folding, pharmacokinetics, and the like. In thepresent invention, it is verified that a bispecific antibody in which ananti-c-Met antibody is fused to an antibody recognizing a secondarytarget, EGFR, or an antigen binding fragment thereof (e.g., scFv) couldimprove and address the stability issue, which was the biggest problemof the pre-existing bispecific antibodies.

One embodiment provides a polypeptide including a novel amino acidsequence. The polypeptide may function as a CDR of an anti-EGFRantibody. In particular, the polypeptide may include one amino acidsequence or a combination of two or more amino acid sequences selectedfrom the group consisting of SEQ ID NO: 109 to SEQ ID NO: 114. Thefunction of the polypeptide including the amino acid sequence of SEQ IDNO: 109 to SEQ ID NO: 114 as a CDR of an anti-EGFR antibody issummarized in Table 1, as follows:

TABLE 1 Heavy chain CDR Light chain CDR CDR- NYDMS CDR- TGSSSNIGNNDVS H1(SEQ ID NO: 109) Ll (SEQ ID NO: 112) CDR- GISHSSGSKYYADSVKG CDR- DDNKRPSH2 (SEQ ID NO: 110) L2 (SEQ ID NO: 113) CDR- KDATPRPLKPFDY CDR-GSWDASLNA H3 (SEQ ID NO: 111) L3 (SEQ ID NO: 114)

In one particular embodiment, the polypeptide may a polypeptideincluding the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 117, apolypeptide including the amino acid sequence of SEQ ID NO: 116 or SEQID NO: 118, or a combination thereof. The polypeptide including theamino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 117, that includesthe amino acid sequences of SEQ ID NOS: 109 to 111, and may have afunction as a heavy chain variable region of an anti-EGFR antibody. Inaddition, the polypeptide including the amino acid sequence of SEQ IDNO: 116 or SEQ ID NO: 118, that includes the amino acid sequences of SEQID NOS: 112 to 114, and may have a function as a light chain variableregion of an anti-EGFR antibody.

<SEQ ID NO: 115, capable of acting as a heavy chain variable region ofan anti-EGFR antibody>

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLE                               CDR-H1WVSGISHSSGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY          CDR-H2YCAKDATPRPLKPFDYWGQGTLVTVSS       CDR-H3

(wherein, the CDRs are marked in bold type)

<SEQ ID NO: 116, capable of acting as a light chain variable region ofan anti-EGFR antibody>

QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVSWYQQLPGTAPKLLI                           CDR-L1YDDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASL  CDR-L2                                  CDR-L3 NAYVFGGGTKLTVLG

(wherein, the CDRs are marked in bold type)

<SEQ ID NO: 117, capable of acting as a heavy chain variable region ofan anti-EGFR antibody>

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKCLEW                               CDR-H1VSGISHSSGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY        CDR-H1CAKDATPRPLKPFDYWGQGTLVTVSS      CDR-H1

(wherein, the CDRs are marked in bold type)

<SEQ ID NO: 118, capable of acting as a heavy chain variable region ofan anti-EGFR antibody>

QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVSWYQQLPGTAPKLLI                           CDR-L1YDDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASL  CDR-L2                                  CDR-L3 NAYVFGCGTKLTVLG

(wherein, the CDRs are marked in bold type)

The polypeptide may act as a precursor or a component of an EGFRantagonist, such as an anti-EGFR antibody, an antigen-binding fragmentthereof, or an anti-EGFR antibody analog (e.g., a peptibody, nanobody,etc.).

Therefore, another embodiment provides an anti-EGFR antagonist includingthe polypeptide. The antagonist inhibits the EGFR activity, and may beone or more selected from the group consisting of an anti-EGFR antibody,an antigen-binding fragment thereof, an anti-EGFR antibody analogue(e.g., a peptibody, nanobody, etc.), and the like.

The term “antagonist” may include any molecule capable of completely orpartially preventing, inhibiting, or neutralizing one or more biologicalactivities of a target (e.g., EGFR). For instance, an antibody as anantagonist may refer to an antibody capable of inhibiting or loweringbiological activities of an antigen (e.g., EGFR) to which the antibodybinds. The antagonist may bind to a receptor for a ligand (target) todecrease receptor phosphorylation, or incapacitating or killing a cellthat is activated by the ligand. In addition, the antagonist maysubstantially decrease an interaction between a receptor and its ligand,by completely blocking the receptor-ligand interaction, binding to thereceptor competitively with its ligand, or modifying or down-regulatingthree-dimensional structure of the receptor.

Term “peptibody (peptide+antibody)” may refer to a fusion proteinwherein a peptide is fused with the whole or a part of a constant regionof an antibody, such as Fc region, and the peptide acts as anantigen-binding region (e.g., a CDR or variable region of a heavy chainand/or light chain), thereby having a structure and functions similar toan antibody.

Term “nanobody” that is also called as a single-domain antibody, mayrefer to an antibody fragment including a single variable domain in amonomeric form and selectively binding to a specific antigen, similarlyto an antibody in a complete form. The nanobody usually has a molecularweight of about 12 kDa to about 15 kDa, which is much smaller than angeneral molecular weight (about 150 kDa to about 160 kDa) of an antibodyin a complete form (including two heavy chains and two light chains),and in some case, smaller than a molecular weight of a Fab fragment or ascFv fragment.

In a particular embodiment, the polypeptide may act as a precursor or acomponent of an anti-EGFR antibody.

Another embodiment provides an anti-EGFR antibody or an antigen-bindingfragment thereof including the polypeptide. The antigen-binding fragmentmay be selected from the group consisting of scFv, (scFv)2, scFv-Fc,Fab, Fab′ and F(ab′)2.

In particular, the anti-EGFR antibody or an antigen-binding fragmentthereof may comprise or consist essentially of:

at least one heavy chain complementarity determining region selectedfrom the group consisting of CDR-H1 including the amino acid sequence ofSEQ ID NO: 109, CDR-H2 including the amino acid sequence of SEQ ID NO:110, and CDR-H3 including the amino acid sequence of SEQ ID NO: 111, ora heavy chain variable region including the at least one heavy chaincomplementarity determining region;

at least one light chain complementarity determining region selectedfrom the group consisting of CDR-L1 including the amino acid sequence ofSEQ ID NO: 112, CDR-L2 including the amino acid sequence of SEQ ID NO:113, and CDR-L3 including the amino acid sequence of SEQ ID NO: 114 or alight chain variable region including the at least one light chaincomplementarity determining region;

a combination of the at least one heavy chain complementaritydetermining region and the at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

For example, the anti-EGFR antibody or an antigen-binding fragmentthereof may comprise or consisting essentially of a heavy chain variableregion including the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO:117, a light chain variable region including the amino acid sequence ofSEQ ID NO: 116 or SEQ ID NO: 118, or a combination thereof.

In a particular embodiment, the anti-EGFR antibody or an antigen-bindingfragment thereof may be an anti-EGFR scFv comprising or consistingessentially of a heavy chain variable region including the amino acidsequence of SEQ ID NO: 115 or SEQ ID NO: 117, and a light chain variableregion including the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO:118.

In the polypeptide or an anti-EGFR scFv comprising or consistingessentially of a heavy chain variable region including the amino acidsequence of SEQ ID NO: 115 or SEQ ID NO: 117, and a light chain variableregion including the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO:118, the heavy chain variable region and the light chain variable regionmay be linked with or without a linker (e.g., a peptide linker). Thepeptide linker may be those including any amino acids of 1 to 100,particularly 2 to 50, and any kinds of amino acids may be includedwithout any restrictions. The peptide linker may include for example,Gly, Asn and/or Ser residues, and also include neutral amino acids suchas Thr and/or Ala. Amino acid sequences suitable for the peptide linkermay be those known in the relevant art. Meanwhile, a length of thepeptide linker may be variously determined within such a limit that thefunctions of the fusion protein will not be affected. For instance, thepeptide linker may be formed by including a total of about 1 to about100, about 2 to about 50, or about 5 to about 25 of one or more selectedfrom the group consisting of Gly, Asn, Ser, Thr, and Ala. In oneembodiment, the peptide linker may be represented as GGGGS_(n) (n is aninteger of about 1 to about 10, particularly an integer of about 2 toabout 5).

As used herein, the term “antibody” may refer to a substance generatedby a stimulation of an antigen in immune system, and there is nospecific limitation in its kinds. The antibody may include all of ananimal antibody, a chimeric antibody, a humanized antibody, and a humanantibody. In addition the antibody may include an antigen-bindingfragment derived from an antibody having an antigen binding affinity.The “complementarity-determining region (CDR)” may refer to a regionwithin a variable region, which give a binding specificity to anantigen. The antigen-binding fragment as described above may be anantibody fragment including at least one complementarity-determiningregion, for example, one or more selected from the group consisting ofscFv, (scFv)2, scFv-Fc, Fab, Fab′, and F(ab′)2.

In the anti-EGFR antibody or an antigen-binding fragment thereof, therest portion of the light chain and the heavy chain portion excludingthe CDRs, the light chain variable region, and the heavy chain variableregion as defined above, e.g., a light chain constant region and a heavychain constant region, may be those from any subtype of immunoglobulin(e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

Based on the ability of specifically binding to EGFR, the anti-EGFRantibody or an antigen-binding fragment thereof may be used in detectingEGFR or confirming activation and/or overproduction (i.e.overexpression) of EGFR.

One embodiment provides a composition for detecting the presence of EGFRincluding the anti-EGFR antibody or an antigen-binding fragment thereof.Another embodiment provides a method of detecting EGFR includingtreating a biological sample with the anti-EGFR antibody or anantigen-binding fragment thereof and detecting an antigen-antibodyreaction (binding). In the method of detecting, when an antigen-antibodyreaction is detected, it can be determined that EGFR is present in thebiological sample. Another embodiment provides a use of the anti-EGFRantibody or an antigen-binding fragment thereof for detecting EGFR. Thebiological sample may be selected from the group consisting of a cell, atissue, a body fluid (e.g., blood, serum, etc.), and the like derivedfrom a mammal including primates such as a human, a monkey, and thelike, or a rodent such as a mouse, a rat, and the like. The biologicalsample may be separated from a living body. The detection of EGFR mayrefer to detection of presence EGFR, expression of EGFR, or the level ofEGFR.

Another embodiment provides a pharmaceutical composition for diagnosingactivation and/or overproduction of EGFR or a disease associated withactivation and/or overproduction of EGFR including the anti-EGFRantibody or an antigen-binding fragment thereof. Another embodimentprovides a method of diagnosing (or determining) activation and/oroverproduction of EGFR or a disease associated with activation and/oroverproduction of EGFR, including treating a biological sample derivedfrom a patient with the anti-EGFR antibody or an antigen-bindingfragment thereof, and measuring a level of an antigen-antibody reaction.In this method, when the level of the antigen-antibody reaction in thebiological sample is higher than that of a normal sample, the patientfrom which the biological sample is derived may be determined as havingactivation and/or overproduction of EGFR or a disease associated withactivation and/or overproduction of EGFR. Therefore, the method mayfurther include treating a normal sample with the anti-EGFR antibody oran antigen-binding fragment thereof, and measuring a level of anantigen-antibody reaction.

The biological sample may be at least one selected from the groupconsisting of a cell, a tissue, fluid (e.g., blood, serum, and the like)and the like, derived from a patient to be diagnosed. The biologicalsample may be separated from a living body. The normal sample may be atleast one selected from the group consisting of a cell, a tissue, fluid(e.g., blood, serum, and the like) and the like, derived from a patienthaving no condition of activation and/or overproduction of EGFR or adisease associated with activation and/or overproduction of EGFR. Thenormal sample may be separated from a living body. The patient may beselected from a mammal, including primates such as a human, a monkey,and the like, and rodents such as a mouse, a rat, and the like.

Another embodiment provides an anti-c-Met/anti-EGFR bispecific antibodyincluding an anti-c-Met antibody or an antigen-binding fragment thereofand an anti-EGFR antibody or an antigen-binding fragment thereof. Theantigen-binding fragment thereof may be selected from the groupconsisting of scFv, (scFv)2, scFvFc, Fab, Fab′, and F(ab′)2.

The “c-Met protein” refers to a receptor tyrosine kinase binding tohepatocyte growth factor. The c-Met proteins may be derived from anyspecies, for example, those derived from primates such as human c-Met(e.g., NP_000236) and monkey c-Met (e.g., Macaca mulatta, NP_001162100),or those derived from rodents such as mouse c-Met (e.g., NP_032617.2)and rat c-Met (e.g., NP_113705.1). The proteins include, for example, apolypeptide encoded by the nucleotide sequence deposited under GenBankAccession Number NM_000245, or a protein encoded by the polypeptidesequence deposited under GenBank Accession Number NM_000236, orextracellular domains thereof. The receptor tyrosine kinase c-Met isinvolved in several mechanisms including cancer incidence, cancermetastasis, cancer cell migration, cancer cell penetration,angiogenesis, etc.

The “EGFR (epidermal growth factor receptor)” is a member of thereceptor tyrosine kinases (RTKs) of HER family. The binding of a ligandto the extracellular domain of EGFR induces receptor homo- or heterodimerization with other ErbB receptors, which in turn results inintracellular self-phosphorylation of specific tyrosine residues. EGFRself-phosphorylation leads to downstream signal transduction networksincluding MAPK and PI3K/Akt activation which affects cell proliferation,angiogenesis and metastasis. Over-expression, gene amplification,mutation, or rearrangement of EGFR are frequently observed in severalhuman malignant tumors and are related to poor prognosis of cancertreatment and bad clinical outcomes. For such reasons, the EGFR becomesan important target in anticancer therapy. The EGFR or HER2 may bederived from mammals, for example, primates such as humans and monkeys,or rodents such as rats and mice. For instance, the EGFR may bepolypeptides encoded by the nucleotide sequences (mRNA) deposited underGenBank Accession Nos. JQ739160, JQ739161, JQ739162, JQ739163, JQ739164,JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1, NM_201282.1, orNM_201283.1.

In one embodiment, the anti-c-Met/anti-EGFR bispecific antibody mayinclude an anti-c-Met antibody or an antigen binding fragment thereof,and an anti-EGFR antibody or an antigen binding fragment thereof whichis linked to the C terminus or N terminus, for example, C terminal, ofthe anti-c-Met antibody or the antigen binding fragment thereof.

In the anti-c-Met/anti-EGFR bispecific antibody, in order to fullyperform the anti-c-Met antibody's activity to mediate intracellularmigration and degradation of c-Met proteins, it may be advantageous thatthe anti-c-Met antibody has its own intact antibody structure. Inaddition, in case of the anti-EGFR antibody, its specific recognitionand binding to EGFR is important, and thus it will be fine that just anantigen-binding fragment recognizing EGFR is included in the bispecificantibody. Therefore, the anti-c-Met/anti-EGFR bispecific antibody may bethose including a complete form of an anti-c-Met antibody (e.g., IgGtype antibody) and an antigen binding fragment of the anti-EGFR antibodylinked to the C terminus of the anti-c-Met antibody.

In the anti-c-Met/anti-EGFR bispecific antibody, the anti-c-Met antibodyor the antigen binding fragment thereof, and the anti-EGFR antibody orthe antigen binding fragment thereof, may be linked via a peptidelinker, or they may be linked directly and without a linker.Furthermore, a heavy chain portion and a light chain portion within theantigen binding fragment, for example, a heavy chain variable region anda light chain variable region within the scFv fragment, may be linkedvia a peptide linker or without a linker. The peptide linker which linksthe anti-c-Met antibody or the antigen binding fragment thereof and theanti-EGFR antibody or the antigen binding fragment thereof, and thepeptide linker which links the heavy chain portion and the light chainportion within the antigen binding fragment, may be identical ordifferent. The peptide linker may be include about 1 to about 100 aminoacid residues, particularly about 2 to about 50, and any kinds of aminoacids may be included without any restrictions. The peptide linker mayinclude for example, Gly, Asn and/or Ser residues, and also includeneutral amino acids such as Thr and/or Ala. Amino acid sequencessuitable for the peptide linker may be those known in the pertinent art.Meanwhile, a length of the peptide linker may be variously determinedwithin such a limit that the functions of the fusion protein will not beaffected. For instance, the peptide linker may be formed by including atotal of about 1 to about 100, about 2 to about 50, or about 5 to about25 of one or more selected from the group consisting of Gly, Asn, Ser,Thr, and Ala. In one embodiment, the peptide linker may be representedas (GGGS)_(n) (n is an integer of about 1 to about 10, particularly aninteger of about 2 to about 5).

In a particular embodiment, the anti-EGFR antibody or an antigen-bidingfragment may comprise or consist essentially of:

at least one heavy chain complementarity determining region selectedfrom the group consisting of CDR-H1 including the amino acid sequence ofSEQ ID NO: 109, CDR-H2 including the amino acid sequence of SEQ ID NO:110, and CDR-H3 including the amino acid sequence of SEQ ID NO: 111 or aheavy chain variable region including the at least one heavy chaincomplementarity determining region;

at least one light chain complementarity determining region selectedfrom the group consisting of CDR-L1 including the amino acid sequence ofSEQ ID NO: 112, CDR-L2 including the amino acid sequence of SEQ ID NO:113, and CDR-L3 including the amino acid sequence of SEQ ID NO: 114 or alight chain variable region including the at least one light chaincomplementarity determining region;

a combination of the at least one heavy chain complementaritydetermining region and the at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

For example, the anti-EGFR antibody or an antigen-binding fragmentthereof may comprise or consist essentially of a heavy chain variableregion including the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO:117, a light chain variable region including the amino acid sequence ofSEQ ID NO: 116 or SEQ ID NO: 118, or a combination thereof.

In a particular embodiment, the anti-EGFR antibody or an antigen-bindingfragment thereof may be an anti-EGFR scFv including a heavy chainvariable region including the amino acid sequence of SEQ ID NO: 115 orSEQ ID NO: 117, and a light chain variable region including the aminoacid sequence of SEQ ID NO: 116 or SEQ ID NO: 118.

The “antigen binding fragment” refers to a fragment of a fullimmunoglobulin structure including parts of the polypeptide including aportion capable of binding to an antigen. For example, it may be scFv,(scFv)₂, Fab, Fab′, or F(ab′)₂, but not be limited thereto. In thepresent invention, the antigen binding fragment may be an antibodyfragment including at least one complementarity determining region, forexample, selected from the group consisting of scFv, (scFv)2, scFv-Fc,Fab, Fab′ and F(ab′)2.

Of the antigen binding fragments, Fab is a structure having variableregions of a light chain and a heavy chain, a constant region of thelight chain, and the first constant region (C_(H1)) of the heavy chain,and it has one antigen binding site.

Fab′ is different from Fab in that it has a hinge region including oneor more cysteine residues at the C-terminal of heavy chain C_(H1)domain. An F(ab′)₂ antibody is formed through disulfide bond of thecysteine residues at the hinge region of Fab′.

Fv is a minimal antibody piece having only a heavy chain variable regionand light chain variable region, and a recombinant technique forproducing the Fv fragment is well known in the pertinent art. Two-chainFv may have a structure in which the heavy chain variable region islinked to the light chain variable region by a non-covalent bond, andsingle-chain Fv (scFv) may generally have a dimer structure as in thetwo-chain Fv in which the variable region of a heavy chain and thevariable region of a light chain are covalently linked via a peptidelinker or they are directly linked to each other at the C-terminalthereof. The peptide linker may be the same as described in the above.

The antigen binding fragments may be obtained using proteases (forexample, a whole antibody is digested with papain to obtain Fabfragments, and is digested with pepsin to obtain F(ab′)₂ fragments), andmay be prepared by a genetic recombinant technique.

In a particular embodiment, the anti-c-Met/anti-EGFR bispecific antibodymay be those including an anti-c-Met antibody, and scFv, (scFv)₂, Fab,Fab′ or F(ab′)₂, for example, scFv, of the anti-EGFR antibody linked tothe C terminus of the anti-c-Met antibody. For instance, scFv, (scFv)₂,Fab, Fab′ or F(ab′)₂ of the anti-EGFR antibody may be those including aheavy chain variable region including the amino acid sequence of SEQ IDNO: 115 or SEQ ID NO: 117 and a light chain variable region includingthe amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 118.

In a particular embodiment, the anti-c-Met/anti-EGFR bispecific antibodymay be those including the anti-c-Met antibody, and scFv, (scFv)₂, Fab,Fab′ or F(ab′)₂ of the anti-EGFR antibody including a heavy chainvariable region including the amino acid sequence of SEQ ID NO: 115 orSEQ ID NO: 117 and a light chain variable region including the aminoacid sequence of SEQ ID NO: 116 or SEQ ID NO: 118, linked to the Cterminal of the anti-c-Met antibody.

The anti-c-Met antibody may be any one recognizing a specific region ofc-Met, e.g., a specific region in the SEMA domain, as an epitope. It maybe any antibody or antigen-binding fragment that acts on c-Met to induceintracellular internalization and degradation of c-Met.

c-Met, a receptor for hepatocyte growth factor(HGF), may be divided intothree portions: extracellular, transmembrane, and intracellular. Theextracellular portion is composed of an α-subunit and a β-subunit whichare linked to each other through a disulfide bond, and contains a SEMAdomain responsible for binding HGF, a PSI domain(plexin-semaphorins-integrin homology domain) and an IPT domain(immunoglobulin-like fold shared by plexins and transcriptional factorsdomain). The SEMA domain of c-Met protein may have the amino acidsequence of SEQ ID NO: 79, and is an extracellular domain that functionsto bind HGF. A specific region of the SEMA domain, that is, a regionincluding the amino acid sequence of SEQ ID NO: 71, which corresponds toa range from amino acid residues 106 to 124 of the amino acid sequenceof the SEMA domain (SEQ ID NO: 79) of c-Met protein, is a loop regionbetween the second and the third propellers within the epitopes of theSEMA domain. The region acts as an epitope for the specific anti-c-Metantibody of the present invention.

The term “epitope” as used herein, refers to an antigenic determinant, apart of an antigen recognized by an antibody. In one embodiment, theepitope may be a region including about 5 or more contiguous(consecutive or non-consecutive) amino acid residues within the SEMAdomain (SEQ ID NO: 79) of c-Met protein, for instance, about 5 to about19 contiguous amino acid residues within the amino acid sequence of SEQID NO: 71. For example, the epitope may be a polypeptide having about 5to about 19 contiguous amino acids selected from among partialcombinations of the amino acid sequence of SEQ ID NO: 71, wherein thepolypeptide essentially includes the amino sequence of SEQ ID NO: 73(EEPSQ) serving as an essential element for the epitope. For example,the epitope may be a polypeptide including, consisting essentially of,or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO:72, or SEQ ID NO: 73.

The epitope including the amino acid sequence of SEQ ID NO: 72corresponds to the outermost part of the loop between the second andthird propellers within the SEMA domain of a c-Met protein. The epitopeincluding the amino acid sequence of SEQ ID NO: 73 is a site to whichthe antibody or antigen-binding fragment according to one embodimentmost specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope whichhas about 5 to about 19 contiguous amino acids selected from amongpartial combinations of the amino acid sequence of SEQ ID NO: 71,including SEQ ID NO: 73 as an essential element. For example, theanti-c-Met antibody may specifically bind to an epitope including theamino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody or an antigen-bindingfragment thereof may comprise or consist essentially of:

at least one heavy chain complementarity determining region (CDR)selected from the group consisting of (a) a CDR-H1 including the aminoacid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the amino acidsequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequence havingabout 8-19 consecutive amino acids within SEQ ID NO: 2 including aminoacid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and(c) a CDR-H3 including the amino acid sequence of SEQ ID NO: 6, SEQ IDNO: 85, or an amino acid sequence having about 6-13 consecutive aminoacids within SEQ ID NO: 85 including amino acid residues from the 1^(st)to 6^(th) positions of SEQ ID NO: 85, or a heavy chain variable regionincluding the at least one heavy chain complementarity determiningregion;

at least one light chain complementarity determining region (CDR)selected from the group consisting of (a) a CDR-L1 including the aminoacid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acidsequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 86, or an amino acid sequencehaving 9-17 consecutive amino acids within SEQ ID NO: 89 including aminoacid residues from the 1^(st) to 9^(th) positions of SEQ ID NO: 89, or alight chain variable region including the at least one light chaincomplementarity determining region;

a combination of the at least one heavy chain complementaritydetermining region and at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectivelyrepresented by following Formulas I to VI, below:

Formula I (SEQ ID NO: 4) Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser,

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II (SEQ ID NO: 5) Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr,

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III (SEQ ID NO: 6) Asp-Asn-Trp-Leu-Xaa₆-Tyr,

wherein Xaa₆ is Ser or Thr,

Formula IV (SEQ ID NO: 7) Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn-Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is Hisor Gln, and Xaa₁₀ is Lys or Asn,

Formula V (SEQ ID NO: 8) Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser orPro, and

Formula VI (SEQ ID NO: 9) Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, orLys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequenceselected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. TheCDR-H2 may include an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may include an aminoacid sequence selected from the group consisting of SEQ ID NOS: 3, 27,28, and 85.

The CDR-L1 may include an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2may include an amino acid sequence selected from the group consisting ofSEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may include an amino acidsequence selected from the group consisting of SEQ ID NOS: 12, 13, 14,15, 16, 37, 86, and 89.

In another embodiment, the antibody or antigen-binding fragment mayinclude

a heavy chain variable region comprising a polypeptide (CDR-H1)including an amino acid sequence selected from the group consisting ofSEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an aminoacid sequence selected from the group consisting of SEQ ID NOS: 2, 25,and 26, and a polypeptide (CDR-H3) including an amino acid sequenceselected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and

a light chain variable region comprising a polypeptide (CDR-L1)including an amino acid sequence selected from the group consisting ofSEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2)including an amino acid sequence selected from the group consisting ofSEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including anamino acid sequence selected from the group consisting of SEQ ID NOS 12,13, 14, 15, 16, 37, 86, and 89.

Animal-derived antibodies produced by immunizing non-immune animals witha desired antigen generally invoke immunogenicity when injected tohumans for the purpose of medical treatment, and thus chimericantibodies have been developed to inhibit such immunogenicity. Chimericantibodies are prepared by replacing constant regions of animal-derivedantibodies that cause an anti-isotype response with constant regions ofhuman antibodies by genetic engineering. Chimeric antibodies areconsiderably improved in an anti-isotype response compared toanimal-derived antibodies, but animal-derived amino acids still havevariable regions, so that chimeric antibodies have side effects withrespect to a potential anti-idiotype response. Humanized antibodies havebeen developed to reduce such side effects. Humanized antibodies areproduced by grafting complementarity determining regions (CDR) whichserve an important role in antigen binding in variable regions ofchimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodiesis choosing the optimized human antibodies for accepting CDRs ofanimal-derived antibodies. Antibody databases, analysis of a crystalstructure, and technology for molecule modeling are used. However, evenwhen the CDRs of animal-derived antibodies are grafted to the mostoptimized human antibody framework, amino acids positioned in aframework of the animal-derived CDRs affecting antigen binding arepresent. Therefore, in many cases, antigen binding affinity is notmaintained, and thus application of additional antibody engineeringtechnology for recovering the antigen binding affinity is necessary.

The anti c-Met antibodies may be mouse-derived antibodies, mouse-humanchimeric antibodies, humanized antibodies, or human antibodies. Theantibodies or antigen-binding fragments thereof may be isolated from aliving body or non-naturally occurring. The antibodies orantigen-binding fragments thereof may be synthetic or recombinant.

An intact antibody includes two full-length light chains and twofull-length heavy chains, in which each light chain is linked to a heavychain by disulfide bonds. The antibody has a heavy chain constant regionand a light chain constant region. The heavy chain constant region is ofa gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, whichmay be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3),gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constantregion is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavychain, and fragments thereof, including a variable region V_(H) thatincludes amino acid sequences sufficient to provide specificity toantigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and ahinge. The term “light chain” refers to a full-length light chain andfragments thereof, including a variable region V_(L) that includes aminoacid sequences sufficient to provide specificity to antigens, and aconstant region C_(L).

The term “complementarity determining region (CDR)” refers to an aminoacid sequence found in a hyper variable region of a heavy chain or alight chain of immunoglobulin. The heavy and light chains mayrespectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1,CDRL2, and CDRL3). The CDR may provide contact residues that play animportant role in the binding of antibodies to antigens or epitopes. Theterms “specifically binding” and “specifically recognized” are wellknown to one of ordinary skill in the art, and indicate that an antibodyand an antigen specifically interact with each other to lead to animmunological activity.

The term “hinge region,” as used herein, refers to a region between CH1and CH2 domains within the heavy chain of an antibody which functions toprovide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1hinge of animal origin is replaced with a human IgG1 hinge or IgG2 hingewhile the disulfide bridges between two heavy chains are reduced fromthree to two in number. In addition, an animal-derived IgG1 hinge isshorter than a human IgG1 hinge. Accordingly, the rigidity of the hingeis changed. Thus, a modification of the hinge region may bring about animprovement in the antigen binding efficiency of the humanized antibody.The modification of the hinge region through amino acid deletion,addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody or an antigen-bindingfragment thereof may be modified by the deletion, insertion, addition,or substitution of at least one amino acid residue on the amino acidsequence of the hinge region so that it exhibit enhanced antigen-bindingefficiency. For example, the antibody may include a hinge regionincluding the amino acid sequence of SEQ ID NO: 100(U7-HC6),101(U6-HC7), 102(U3-HC9), 103(U6-HC8), or 104(U8-HC5), or a hinge regionincluding the amino acid sequence of SEQ ID NO: 105 (non-modified humanhinge). In particular, the hinge region has the amino acid sequence ofSEQ ID NO: 100 or 101.

In one embodiment, the anti-c-Met antibody or antigen-binding fragmentmay include a variable region of the heavy chain including the aminoacid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94, avariable region of the light chain including the amino acid sequence ofSEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107, or acombination thereof.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody.The monoclonal antibody may be produced by the hybridoma cell linedeposited with Accession No. KCLRF-BP-00220, which binds specifically tothe extracellular region of c-Met protein (refer to Korean PatentPublication No. 2011-0047698, the disclosure of which is incorporated inits entirety herein by reference). The anti-c-Met antibody may includeall the antibodies defined in Korean Patent Publication No.2011-0047698.

In the anti-c-Met antibody, the rest portion of the light chain and theheavy chain portion excluding the CDRs, the light chain variable region,and the heavy chain variable region as defined above, that is the lightchain constant region and the heavy chain constant region, may be thosefrom any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1,IgG2, IgG3, IgG4), IgM, and the like).

By way of further example, the anti-c-Met antibody or the antibodyfragment may include:

a heavy chain including the amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 62 (wherein theamino acid sequence from amino acid residues from the 1^(st) to 17^(th)positions is a signal peptide), or the amino acid sequence from the18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequenceof SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to17^(th) positions is a signal peptide), the amino acid sequence from the18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequenceof SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to17^(th) positions is a signal peptide), and the amino acid sequence fromthe 18^(th) to 460^(th) positions of SEQ ID NO: 66; and

a light chain including the amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 68 (wherein theamino acid sequence from the 1^(st) to 20^(th) positions is a signalpeptide), the amino acid sequence from the 21^(st) to 240^(th) positionsof SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein theamino acid sequence from the 1^(st) to 20^(th) positions is a signalpeptide), the amino acid sequence from the 21^(st) to 240^(th) positionsof SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the groupconsisting of:

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 108.

According to an embodiment, the anti-c-Met antibody may include a heavychain including the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the sequence fromthe 21^(st) to 240^(th) positions of SEQ ID NO: 68, or a heavy chainincluding the amino acid sequence from the 18^(th) to 460^(th) positionsof SEQ ID NO: 66 and a light chain including the sequence of SEQ ID NO:108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa(κ) constant region, and the polypeptide with the amino acid sequence ofSEQ ID NO: 68 is a polypeptide obtained by replacing histidine atposition 62 (corresponding to position 36 of SEQ ID NO: 68 according tokabat numbering) of the polypeptide with the amino acid sequence of SEQID NO: 70 with tyrosine. The production yield of the antibodies may beincreased by the replacement. The polypeptide with the amino acidsequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serineat position 32 of SEQ ID NO: 108 (corresponding to position 52 of SEQ IDNO: 68, which corresponds to position 27e according to kabat numberingin the amino acid sequence from amino acid residues 21 to 240 of SEQ IDNO: 68; positioned within CDR-L1) with tryptophan. By such replacement,antibodies and antibody fragments including such sequences exhibitsincreased activities, such as c-Met biding affinity, c-Met degradationactivity, Akt phosphorylation inhibition, and the like.

In another embodiment, the anti-c-Met antibody may include a light chaincomplementarity determining region including the amino acid sequence ofSEQ ID NO: 106, a light chain variable region including the amino acidsequence of SEQ ID NO: 107, or a light chain including the amino acidsequence of SEQ ID NO: 108.

The anti-c-Met/anti-EGFR bispecific antibody can not only inhibit theactivity of c-Met and EGFR by the internalization and degradationactivity of anti-c-Met antibody but also fundamentally block them byreducing the total amounts of c-Met and EGFR by the degradation thereof.Accordingly, the anti-c-Met/anti-EGFR bispecific antibody can obtainefficient effects even when applied to patients who have developedresistance against pre-existing anti-EGFR antibodies.

Another embodiment provides a pharmaceutical composition including theanti-EGFR antibody or an antigen-binding fragment thereof as an activeingredient. Another embodiment provides a pharmaceutical compositionincluding the anti-c-Met/anti-EGFR bispecific antibody as an activeingredient.

In particular, another embodiment provides a pharmaceutical compositionfor preventing and/or treating a cancer including the anti-EGFR antibodyor an antigen-binding fragment thereof as an active ingredient. Anotherembodiment provides a pharmaceutical composition for preventing and/ortreating a cancer including the anti-c-Met/anti-EGFR bispecific antibodyas an active ingredient.

Another embodiment provides a method of prevention and/or treatment acancer, including administering a pharmaceutical effective amount of theanti-EGFR antibody or an antigen-binding fragment thereof to a patientin need of the prevention and/or treatment of the cancer. Anotherembodiment provides a method of prevention and/or treatment a cancer,including administering a pharmaceutical effective amount of theanti-c-Met/anti-EGFR bispecific antibody to a patient in need of theprevention and/or treatment of the cancer. The method of preventionand/or treatment a cancer may further comprises a step of identifyingthe patient in need of the prevention and/or treatment of the cancer,prior to the step of administering.

The cancer may be any cancer associated with overexpression and/orabnormal activation of c-Met and/or EGFR. The cancer may be any cancerin which EGFR and/or c-Met possibly plays an important role forproliferation, invasion, and metastasis, including the resistant cancersto EGFR therapy. The cancer may be a solid cancer or hematologicalcancer and for instance, may be, but not limited to, one or moreselected from the group consisting of squamous cell carcinoma,small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma ofthe lung, squamous cell carcinoma of the lung, peritoneal carcinoma,skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer nearthe anus, esophagus cancer, small intestinal tumor, endocrine glandcancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma,urethral cancer, chronic or acute leukemia, lymphocytic lymphoma,hepatoma, gastric cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatocellularadenoma, breast cancer, colon cancer, large intestine cancer,endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidneycancer, prostate cancer, vulvar cancer, thyroid cancer, head or neckcancer, brain cancer, and the like. In particular, the cancer may becancer having resistance against pre-existing anticancer drugs, forexample, antagonists against EGFR and/or antagonists against c-Met. Theprevention and/or treatment effects of the cancers may include effectsof not only suppressing the growth of the cancer cells but alsosuppressing deterioration of cancers due to migration, invasion, and/ormetastasis thereof. Therefore, the curable cancers may include bothprimary cancers and metastatic cancers. Thus, the pharmaceuticalcomposition or method may be for preventing and/or treating cancermetastasis.

In the pharmaceutical composition or method, the pharmaceuticallyeffective amount of the anti-EGFR antibody or an antigen-bindingfragment thereof or the anti-c-Met/anti-EGFR bispecific antibody may beadministered along with at least one additive selected from the groupconsisting of a pharmaceutically acceptable carriers, diluents, andexcipients.

The pharmaceutically acceptable carrier to be included in thecomposition may be those commonly used for the formulation ofantibodies, which may be one or more selected from the group consistingof lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia,calcium phosphate, alginates, gelatin, calcium silicate,micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate,talc, magnesium stearate, and mineral oil, but are not limited thereto.The pharmaceutical composition may further include one or more selectedfrom the group consisting of a lubricant, a wetting agent, a sweetener,a flavor enhancer, an emulsifying agent, a suspension agent, andpreservative.

The pharmaceutical composition or the anti-EGFR antibody or anantigen-binding fragment thereof or the anti-c-Met/anti-EGFR bispecificantibody may be administered orally or parenterally. The parenteraladministration may include intravenous injection, subcutaneousinjection, muscular injection, intraperitoneal injection, endothelialadministration, local administration, intranasal administration,intrapulmonary administration, and rectal administration. Since oraladministration leads to digestion of proteins or peptides, an activeingredient in the compositions for oral administration must be coated orformulated to prevent digestion in stomach. In addition, thecompositions may be administered using an optional device that enablesan active substance to be delivered to target cells.

A suitable dosage of the pharmaceutical composition, the anti-EGFRantibody or an antigen-binding fragment thereof, or theanti-c-Met/anti-EGFR bispecific antibody may be prescribed in a varietyof ways, depending on factors such as formulation methods,administration methods, age of patients, body weight, gender, pathologicconditions, diets, administration time, administration route, excretionspeed, and reaction sensitivity. A desirable dosage of thepharmaceutical composition or the anti-c-Met/anti-EGFR bispecificantibody may be in the range of about 0.001 to 100 mg/kg for an adult.For example, the suitable dosage of the pharmaceutical composition, theanti-EGFR antibody or an antigen-binding fragment thereof, or theanti-c-Met/anti-EGFR bispecific antibody may be about 0.001 to about1000 mg/kg, about 0.01 to about 100 mg/kg, or 0.1 to 50 mg/kg, per aday, but not be limited thereto. The term “pharmaceutically effectiveamount” used herein refers to an amount of the active ingredient (i.e.,the anti-EGFR antibody or an antigen-binding fragment thereof, or theanti-c-Met/anti-EGFR bispecific antibody) exhibiting effects inpreventing or treating cancer, and may be properly determined in avariety of ways, depending on factors such as formulation methods,administration methods, age of patients, body weight, gender, pathologicconditions, diets, administration time, administration route, excretionspeed, and reaction sensitivity.

The pharmaceutical composition or the anti-c-Met/anti-EGFR bispecificantibody may be formulated with a pharmaceutically acceptable carrierand/or excipient into a unit or a multiple dosage form by a methodeasily carried out by a skilled person in the pertinent art. The dosageform may be a solution in oil or an aqueous medium, a suspension, syrup,an emulsifying solution, an extract, powder, granules, a tablet, or acapsule, and may further include a dispersing or a stabilizing agent.

In addition, the pharmaceutical composition or the anti-c-Met/anti-EGFRbispecific antibody may be administered as an individual drug, ortogether with other drugs, and may be administered sequentially orsimultaneously with pre-existing drugs.

Since the pharmaceutical composition includes an antibody or an antigenbinding fragment thereof, it may be formulated as an immunoliposome. Theliposome containing an antibody may be prepared using a well-knownmethod in the pertinent art. The immunoliposome is a lipid compositionincluding phosphatidylcholine, cholesterol, andpolyethyleneglycol-derivatized phosphatidylethanolamine, and may beprepared by a reverse phase evaporation method. For example, Fab′fragments of an antibody may be conjugated to the liposome through adisulfide exchange reaction. A chemical drug such as doxorubicin may beadditionally included in the liposome.

The subject to which the pharmaceutical composition is administered orthe patient to which the prevention and/treatment method is applied maybe mammals, for example, primates such as humans and monkeys, or rodentssuch as rats and mice, but are not be limited thereto. The subject orthe patient may be a cancer patient having resistance againstpre-existing anticancer drugs, for example, antagonists against thetarget cell membrane proteins (e.g., EGFR).

Another embodiment provides a polynucleotide encoding a polypeptideincluding one amino acid sequence or a combination of two or more aminoacid sequences selected from the group consisting of SEQ ID NO: 109 toSEQ ID NO: 114. In a particular embodiment, the polynucleotide mayencode a polypeptide including the amino acid sequence of SEQ ID NO: 115or SEQ ID NO: 117, a polypeptide including the amino acid sequence ofSEQ ID NO: 116 or SEQ ID NO: 118, or a combination thereof. Anotherembodiment provides a recombinant vector including the polynucleotide.Another embodiment provides a recombinant cell transfected with therecombinant vector.

The term “vector” used herein refers to a means for expressing a targetgene in a host cell. For example, it includes a plasmid vector, a cosmidvector, and a virus vector such as a bacteriophage vector, an adenovirusvector, a retrovirus vector and an adeno-associated virus vector.Suitable recombinant vectors may be constructed by manipulating plasmidsoften used in the art (for example, pSC101, pGV1106, pACYC177, ColE1,pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14,pGEX series, pET series, pUC19, and the like), a phage (for example,λgt4λB, λ-Charon, λΔz1, M13, and the like), or a virus (for example,SV40, and the like), but not be limited thereto.

In the recombinant vector, the polynucleotides may be operatively linkedto a promoter. The term “operatively linked” used herein refers to afunctional linkage between a nucleotide expression regulating sequence(for example, a promoter sequence) and other nucleotide sequences. Thus,the regulating sequence may regulate the transcription and/ortranslation of the other nucleotide sequences by being operativelylinked.

The recombinant vector may be constructed for cloning or expression. Theexpression vector may be any ordinary vectors known in the pertinent artfor expressing an exogenous protein in plants, animals, ormicroorganisms. The recombinant vector may be constructed using variousmethods known in the art.

The recombinant vector may be constructed using a prokaryotic cell or aeukaryotic cell as a host. For example, when a prokaryotic cell is usedas a host cell, the expression vector used generally includes a strongpromoter capable of initiating transcription (for example, pL^(λ)promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7promoter, and the like), a ribosome binding site for initiatingtranslation, and a transcription/translation termination sequence. Whena eukaryotic cell is used as a host cell, the vector used generallyincludes the origin of replication acting in the eukaryotic cell, forexample, an f1 replication origin, a SV40 replication origin, a pMB1replication origin, an adeno replication origin, an AAV replicationorigin, or a BBV replication origin, but is not limited thereto. Apromoter in an expression vector for a eukaryotic host cell may be apromoter derived from the genomes of mammalian cells (for example, ametallothionein promoter, and the like) or a promoter derived frommammalian viruses (for example, an adenovirus late promoter, a vacciniavirus 7.5K promoter, a SV40 promoter, a cytomegalovirus promoter, a tkpromoter of HSV, and the like). A transcription termination sequence inan expression vector for a eukaryotic host cell may be, in general, apolyadenylation sequence.

The recombinant cell may be those obtained by transfecting therecombinant vector into a suitable host cell. Any host cells known inthe pertinent art to enable stable and continuous cloning or expressionof the recombinant vector may be used as the hose cell. Suitableprokaryotic host cells may be one or more selected from E. coli JM109,E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E.coli W3110, Bacillus species strains such as Bacillus subtillis, orBacillus thuringiensis, intestinal bacteria and strains such asSalmonella typhymurum, Serratia marcescens, and various Pseudomonasspecies. Suitable eukaryotic host cells to be transformed may be one ormore selected from yeasts, such as Saccharomyces cerevisiae, insectcells, plant cells, and animal cells, for example, Sp2/0, Chinesehamster ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2,Huh7, 3T3, RIN, and MDCK cell lines, but not be limited thereto.

The polynucleotide or the recombinant vector including the same may betransferred (transfected) into a host cell by using known transfermethods. Suitable transfer methods for prokaryotic host cells mayinclude a method using CaCl₂ and electroporation. Suitable transfermethods for eukaryotic host cells may include microinjection, calciumphosphate precipitation, electroporation, liposome-mediatedtransfection, and gene bombardment, but are not limited thereto.

A transformed host cell may be selected using a phenotype expressed by aselected marker by any methods known in the art. For example, if theselected marker is a gene that is resistant to a specific antibiotic, atransformant may be easily selected by being cultured in a mediumincluding the antibiotic.

The anti-c-Met/anti-EGFR bispecific antibody may be expected to exhibitan improved effect compared to a previously known anti-c-Met antibody,as follows:

1. an increased cancer cell inhibition effect compared to pre-existinganti-c-Met antibodies or EGFR-targeting drugs.

2. a cancer cell inhibition effect on a cancer cells having resistanceto pre-existing anti-c-Met antibodies or EGFR-targeting drugs.

Hereafter, the present invention will be described in detail byexamples.

The following examples are intended merely to illustrate the inventionand are not construed to restrict the invention.

EXAMPLES Reference Example 1 Construction of Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridomacell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old,was intraperitoneally injected with a mixture of 100 μg of humanc-Met/Fc fusion protein (R&D Systems) and one volume of completeFreund's adjuvant. Two weeks after the injection, a secondintraperitoneal injection was conducted on the same mice with a mixtureof 50 μg of human c-Met/Fc protein and one volume of incomplete Freund'sadjuvant. One week after the second immunization, the immune responsewas finally boosted. Three days later, blood was taken from the tails ofthe mice and the sera were 1/1000 diluted in PBS and used to examine atiter of antibody to c-Met by ELISA. Mice found to have a sufficientantibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) wereimmunized with an intraperitoneal injection of a mixture of 50 μg ofhuman c-Met/Fc fusion protein and one volume of PBS. The immunized micewere anesthetized before excising the spleen from the left half of thebody. The spleen was meshed to separate splenocytes which were thensuspended in a culture medium (DMEM, GIBCO, Invitrogen). The cellsuspension was centrifuged to recover the cell layer. The splenocytesthus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸cells), followed by spinning to give a cell pellet. The cell pellet wasslowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) inDMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To thecells was added 10 mL of DMEM over 10 min, after which incubation wasconducted in a water bath at 37° C. for 5 min. Then the cell volume wasadjusted to 50 mL before centrifugation. The cell pellet thus formed wasresuspended at a density of 1-2×10⁵ cells/mL in a selection medium (HATmedium) and 0.1 mL of the cell suspension was allocated to each well of96-well plates which were then incubated at 37° C. in a CO₂ incubator toestablish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies toc-Met Protein

From the hybridoma cell population established in Reference Example1.1.2, hybridoma cells which showed a specific response to c-Met proteinwere screened by ELISA using human c-Met/Fc fusion protein and human Fcprotein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2μg/mL)/well to microtiter plates and allowed to adhere to the surface ofeach well. The antibody that remained unbound was removed by washing.For use in selecting the antibodies that do not bind c-Met but recognizeFc, human Fc protein was attached to the plate surface in the samemanner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was addedin an amount of 50 μL to each well of the plates and incubated for 1hour. The cells remaining unreacted were washed out with a sufficientamount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouseIgG-horseradish peroxidase (HRP) was added to the plates and incubatedfor 1 hour at room temperature. The plates were washed with a sufficientamount of TBST, followed by reacting the peroxidase with a substrate(OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically andstrongly bind to human c-Met but not human Fc were selected repeatedly.From the hybridoma cell lines obtained by repeated selection, a singleclone producing a monoclonal antibody was finally separated by limitingdilution. The single clone of the hybridoma cell line producing themonoclonal antibody was deposited with the Korean Cell Line ResearchFoundation, an international depository authority located atYungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, with AccessionNo. KCLRF-BP-00220 according to the Budapest Treaty (refer to KoreanPatent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was culturedin a serum-free medium, and the monoclonal antibody (AbF46) was producedand purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM)supplemented with 10% (v/v) FBS were centrifuged and the cell pellet waswashed twice or more with 20 mL of PBS to remove the FBS therefrom.Then, the cells were resuspended in 50 mL of DMEM and incubated for 3days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant wasstored at 4° C. before use or immediately used for the separation andpurification of the antibody. An AKTA system (GE Healthcare) equippedwith an affinity column (Protein G agarose column; Pharmacia, USA) wasused to purify the antibody from 50 to 300 mL of the supernatant,followed by concentration with an filter (Amicon). The antibody in PBSwas stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solvethis problem, chAbF46, a chimeric antibody, was constructed from themouse antibody AbF46 produced in Experimental Example 1.1.4 by replacingthe constant region, but not the variable region responsible forantibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequenceof “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for aheavy chain and the nucleotide sequence of “EcoRI-signalsequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain andsynthesized. Then, a DNA fragment having the heavy chain nucleotidesequence (SEQ ID NO: 38) and a DNA fragment having the light chainnucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB,R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TACloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no.12762-019, Invitrogen), and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no.8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA: light chain DNA) and mixed with 2 ml of OptiPro™SFM (Invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v)FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-freeDMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify a chimeric antibody AbF46 (hereinafterreferred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric AntibodychAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes whichshare the highest identity/homology with the VH gene of the mouseantibody AbF46 purified in Reference Example 1.2 were analyzed. An IgBLAST (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has anidentity/identity/homology of 83% at the amino acid level. CDR-H1,CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according toKabat numbering. A design was made to introduce the CDR of the mouseantibody AbF46 into the framework of VH3-71. Back mutations to the aminoacid sequence of the mouse AbF46 were conducted at positions 30 (S→T),48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated atpositions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ IDNO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzedby a BLAST search. The result revealed that the VH3 subtype, known to bemost stable, is very similar in framework and sequence to the mouseantibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46were defined according to Kabat numbering and introduced into the VH3subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO:44), human germline genes which share the highest identity/homology withthe VH gene of the mouse antibody AbF46 were analyzed. An Ig BLASTsearch result revealed that VK4-1 has a identity/homology of 75% at theamino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46were defined according to Kabat numbering. A design was made tointroduce the CDR of the mouse antibody AbF46 into the framework ofVK4-1. Back mutations to the amino acid sequence of the mouse AbF46 wereconducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one backmutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share thehighest identity/homology with the VL gene of the mouse antibody AbF46were analyzed by a search for BLAST. As a result, VK2-40 was selected.VL and VK2-40 of the mouse antibody AbF46 were found to have aidentity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, andCDR-L3 of the mouse antibody were defined according to Kabat numberingand introduced into the framework of VK4-1. Back mutations wereconducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworkswere analyzed. A Blast search revealed that the Vk1 subtype, known to bethe most stable, is very similar in framework and sequence to the mouseantibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46were defined according to Kabat numbering and introduced into the Vk1subtype. Back mutations were conducted at positions 36 (Y→H), 46 (L→M),and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences(H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO:49) and DNA fragments having the light chain nucleotide sequences(H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO:52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) andXhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kitenclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019,Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01),respectively, so as to construct recombinant vectors for expressing ahumanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA: light chain DNA) and mixed with 2 ml of OptiPro™SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify a humanized antibody AbF46 (hereinafterreferred to as “huAbF46”). The humanized antibody huAbF46 used in thefollowing examples included a combination of H4-heavy (SEQ ID NO: 42)and H4-light (SEQ ID NO: 46).

1.4. Construction of scFv Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavyand light chain variable regions of the huAbF46 antibody, a gene wasdesigned to have the structure of “VH-linker-VL” for each of the heavyand the light chain variable region, with the linker including the aminoacid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). Apolynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv ofhuAbF46 was synthesized in Bioneer and an expression vector for thepolynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, sixcomplementary determining regions (CDRs) were defined according to Kabatnumbering. The CDRs are given in Table 2, below.

TABLE 2 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2FIRNKANGYTTEYSASVKG(SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of theantibody, primers were designed as follows. Conventionally, N codonswere utilized to introduce bases at the same ratio (25% A, 25% G, 25% C,25% T) into desired sites of mutation. In this experiment, theintroduction of random bases into the CDRs of huAbF46 was conducted insuch a manner that, of the three nucleotides per codon in the wild-typepolynucleotide encoding each CDR, the first and second nucleotidesconserved over 85% of the entire sequence while the other threenucleotides were introduced at the same percentage (each 5%) and thatthe same possibility was imparted to the third nucleotide (33% G, 33% C,33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity forc-Met

The construction of antibody gene libraries through the introduction ofrandom sequences was carried out using the primers synthesized in thesame manner as in Reference Example 1.5.1. Two PCR products wereobtained using a polynucleotide covering the scFV of huAbF46 as atemplate, and were subjected to overlap extension PCR to give scFvlibrary genes for huAbF46 antibodies in which only desired CDRs weremutated. Libraries targeting each of the six CDRs prepared from the scFVlibrary genes were constructed.

The affinity for c-Met of each library was compared to that of thewildtype. Most libraries were lower in affinity for c-Met, compared tothe wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met,the nucleotide sequence of scFv from each clone was analyzed. Thenucleotide sequences thus obtained are summarized in Table 3 and wereconverted into IgG forms. Four antibodies which were respectivelyproduced from clones L3-1, L3-2, L3-3, and L3-5 were used in thesubsequent experiments.

TABLE 3 Library Clone onstructed CDR Sequence H11-4 CDR-H1PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27)YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34)L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15)L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selectedantibodies were designed to have the structure of “EcoRI-signalsequence-VH-NheI—CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46antibodies were used as they were because their amino acids were notchanged during affinity maturation. In the case of the hinge region,however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of thehinge of human IgG1. Genes were also designed to have the structure of“EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain.Polypeptides encoding light chain variable regions of the fourantibodies which were selected after the affinity maturation weresynthesized in Bioneer. Then, a DNA fragment having the heavy chainnucleotide sequence (SEQ ID NO: 38) and DNA fragments having the lightchain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3:SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO:59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNAfragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digestedwith EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into apOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody ExpressKit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit(Cat no. 8300-01), respectively, so as to construct recombinant vectorsfor expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA: light chain DNA) and mixed with 2 ml of OptiPro™SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify four affinity-matured antibodies(hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2(L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-SubstitutedhuAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7,huAbF46-H4-A1 was found to be the highest in affinity for c-Met and thelowest in Akt phosphorylation and c-Met degradation degree. In theantibody, the hinge region, or the constant region and the hinge region,were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chainincluding the heavy chain variable region of huAbF46-H4-A1, U6-HC7hinge, and the constant region of human IgG1 constant region, and alight chain including the light chain variable region of huAbF46-H4-A1and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge)was composed of a heavy chain including a heavy chain variable region, ahuman IgG2 hinge region, and a human IgG1 constant region, and a lightchain including the light chain variable region of huAbF46-H4-A1 and ahuman kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) wascomposed of the heavy chain variable region of huAbF46-H4-A1, a humanIgG2 hinge region, and a human IgG2 constant region, and a light chainincluding the light variable region of huAbF46-H4-A1 and a human kappaconstant region. The histidine residue at position 36 on the human kappaconstant region of the light chain was changed to tyrosine in all of thethree antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ IDNO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavychain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and ahuman IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding apolypeptide (SEQ ID NO: 64) composed of the heavy chain variable regionof huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, apolynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66)composed of the heavy chain variable region of huAbF46-H4-A1, a humanIgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the lightchain variable region of huAbF46-H4-A1, with a tyrosine residue insteadof histidine at position 36, and a human kappa constant region weresynthesized in Bioneer. Then, the DNA fragments having heavy chainnucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kitenclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019,Invitrogen) while DNA fragments having light chain nucleotide sequenceswere inserted into a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) soas to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle™ MAX reagent (invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE healthcare, 17-0405-03), followed by elutionwith IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to finally purify three antibodies (huAbF46-H4-A1(U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)).Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was selected for thefollowing examples, and name as L3-1Y-IgG2.

Example 1 Preparation of Anti-EGFR scFv

An anti-EGFR scFv binding to EGFR was prepared by inserting a peptidelinker of (GGGGS)₃ between a heavy chain variable region of SEQ ID NO:115 and a light chain variable region of SEQ ID NO: 116. In particular,the DNA sequence encoding a (GGGGS)₃ linker peptide was added to the DNAsequence (SEQ ID NO: 119) encoding the heavy chain variable region (SEQID NO: 115) and the DNA sequence (SEQ ID NO: 120) encoding the lightchain variable region (SEQ ID NO: 116) of a humanized anti-EGFR antibodyusing an automatic gene synthesis (Bioneer Inc.) to synthesize a DNAfragment encoding a scFv of the anti-EGFR antibody. An anti-EGFR scFvprepared from the synthesized DNA fragment was named as “anti-EGFRantibody E-2”.

The amino acid sequences of the heavy chain variable region and thelight chain variable region of the prepared anti-EGFR scFv, and codingnucleotide sequences thereof are summarized in Table 4, as follows(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-H1,CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3, in sequence):

TABLE 4 Heavy chain variable region of Light chain variable region ofanti-EGFR antibody E-2 anti-EGFR antibody E-2 Amino EVQLLESGGGLVQPGGSLRLQSVLTQPPSASGTPGQRVTISC acid SCAASGFTFSNYDMSWVRQ TGSSSNIGNNDVSWYQQLPGsequence APGKGLEWVSGISHSSGSKY TAPKLLIYDDNKRPSGVPDRF YADSVKGRFTISRDNSKNTLSGSKSGTSASLAISGLRSEDEA YLQMNSLRAEDTAVYYCAK DYYCGSWDASLNAYVFGGGDATPRPLKPFDYWGQGTLV TKLTVLG (SEQ ID NO: 116) TVSS (SEQ ID NO: 115)Coding GAGGTGCAGCTGTTGGAGT CAGTCTGTGCTGACTCAGCC nucleotideCTGGGGGAGGCTTGGTACA ACCCTCAGCGTCTGGGACCC sequence GCCTGGGGGGTCCCTGAGACCGGGCAGAGGGTCACCATC CTCTCCTGTGCAGCCTCTGG TCTTGTACTGGCTCTTCATCATTCACCTTTAGCAATTAT TAATATTGGCAATAATGAT GATATGAGCTGGGTCCGCCGTCTCCTGGTACCAGCAGCT AGGCTCCAGGGAAGGGGCT CCCAGGAACGGCCCCCAAACGGAGTGGGTCTCAGGGAT TCCTCATCTATGATGATAAT CTCTCATAGTAGTGGTAGAAGCGGCCAAGCGGGGTCC TAAATATTACGCTGATTC CTGACCGATTCTCTGGCTCCTGTAAAAGGTCGGTTCACC AAATCTGGCACCTCAGCCTC ATCTCCAGAGACAATTCCACCTGGCCATCAGTGGGCTCC AGAACACGCTGTATCTGCA GGTCCGAGGATGAGGCTGATAATGAACAGCCTGAGAGCC TATTACTGTGGTTCTTGGGA GAGGACACGGCCGTGTATTTGCTAGCCTGAATGCTTAT ACTGTGCGAAAGATGCTA GTCTTCGGCGGAGGCACCAACTCCGCGTCCGCTGAAGC GCTGACGGTCCTAGGC (SEQ CTTTCGACTACTGGGGCCA ID NO: 120)GGGTACACTGGTCACCGTG AGCTCA (SEQ ID NO: 119)

A modified anti-EGFR scFv (heavy chain variable region: SEQ ID NO: 117and light chain variable region: SEQ ID NO: 118) was prepared asdescribed above, with the exception that the amino acid, G, at 44^(th)position of the heavy chain variable region (SEQ ID NO: 115) wassubstituted with C, and the amino acid, G, at 100^(th) position of thelight chain variable region (SEQ ID NO: 116) was substituted with C. Theamino acid location within the antibody complies with kabat numberingsystem. Such modifications (substitutions) can increase the stability ofthe anti-EGFR scFv.

<SEQ ID NO: 117: heavy chain variable region of modified anti-EGFRantibody E-2>

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKCLEWVSGISHSSGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDATPRPLKPFDYWGQGTLVTVSS

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-H1,CDR-H2, and CDR-H3, in sequence)

<SEQ ID NO: 118: light chain variable region of modified anti-EGFRantibody E-2>

QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVSWYQQLPGTAPKLLIYDDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDASLNAYV FGCGTKLTVLG

(wherein the sequences marked in bold type indicate CDRs, i.e., CDR-L1,CDR-L2, and CDR-L3, in sequence)

The thus obtained modified anti-EGFR scFv (including SEQ ID NO: 117 andSEQ ID NO: 118) was used to manufacture the following bispecificantibodies.

Example 2 Preparation of Anti-c-Met/Anti-EGFR Bispecific Antibody

The modified anti-EGFR scFv (including SEQ ID NO: 117 and SEQ ID NO:118) prepared in the above Example 1 was fused at the c-terminal of Fcof the anti-c-Met antibody L3-1Y-IgG2 prepared in the above referenceexample 1. The fusion procedures are as follows.

A DNA segment having a base sequence (SEQ ID NO: 66) corresponding tothe heavy chain of the anti-c-Met antibody L3-1Y-IgG2 prepared in abovereference example 1 was inserted into a pcDNA™3.3-TOPO TA Cloning Kit(Cat no. 8300-01) which is included in OptiCHO™ Antibody Express Kit(Cat no. 12762-019) by Invitrogen Inc., and a DNA segment having a basesequence (SEQ ID NO: 68) corresponding to the light chain of theanti-c-Met antibody L3-1Y-IgG2 was inserted into a pOptiVEC™-TOPO TACloning Kit. Thereafter, the anti-EGFR scFv coding DNA prepared inExample 1 was fused at the c-terminal of Fc of L3-1Y-IgG2 inserted intopcDNA™3.3, using the coding DNA sequence of a linker peptide having 10amino acid lengths consisting of (G4S)2, to construct vectors for theexpression of bispecific antibodies.

The constructed vectors were each amplified using Qiagen Maxiprep kit(Cat no. 12662 and their temporary expressions were performed usingFreestyle™ MAX 293 Expression System (invitrogen). A cell line used was293 F cells, which were cultured in a suspension culture manner usingFreeStyle™ 293 Expression Medium as a medium. One day before thetemporary expression, the cells were prepared at a concentration of5×10⁵ cells/ml and after 24 hours, their temporary expression startedwhen the number of the cells reached 1×10⁶ cells/ml. Transfection wasperformed by a liposomal reagent method using Freestyle™ MAX reagent(Invitrogen). DNA was prepared in a 15-ml tube in a ratio of heavy chainDNA:light chain DNA=3:2 and mixed with 2 ml of OptiPro™ SFM (invtrogen)(A), and 100 μl of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM weremixed in another 15-ml tube (B), and after (A) and (B) were mixed andincubated for 15 min., the mixture solution was then slowly mixed intothe cells which were prepared one day before. After the transfection wascomplete, the cells were cultured in a 37° C., 80% humidity, 8% CO₂, 130rpm incubator for 5 days.

The cultured cells were centrifuged to obtain each 100 ml ofsupernatants, which were then purified using AKTA Prime (GE healthcare).The culture was flowed at a flow rate of 5 ml/min. onto the AKTA Primeinstalled with Protein A column (GE healthcare, 17-0405-03) to performelution using an IgG elution buffer (Thermo Scientific, 21004). Thebuffer was replaced by a PBS buffer to finally obtain purifiedbispecific anti-c-Met/anti-EGFR antibodies.

The thus prepared anti-c-Met/anti-EGFR bispecific antibody in which themodified anti-EGFR scFv is fused at the c-terminal of L3-1Y-IgG2 wasnamed ME22S.

Example 3 Properties of Anti-c-Met/Anti-EGFR Bispecific Antibody

To examine the property of the anti-c-Met/anti-EGFR bispecific antibodyME22S prepared in Example 2, 20 ug of ME22S was injected into HPLCsystem (WATERS 2695) equipped with TSKG3000SWXL column (Tosho) at thevelocity of 0.5 ml/min, to conduct Size Exclusion Chromatography byHPLC.

The obtained results are shown in FIG. 2. As shown in FIG. 2, theanti-c-Met/anti-EGFR bispecific antibody ME22S forms a slight amount ofsoluble dimer (<1%)m, indicating that it is very stable molecule.

Example 4 Dual Binding of Anti-c-Met/Anti-EGFR Bispecific Antibody

To examine whether the anti-c-Met/anti-EGFR bispecific antibodyrecognizes both of the antigens, c-Met and EGFR, the experimentationusing Biacore T100 (GE) was performed. An anti-6xHis antibody (#MAB050,R&D Systems) was immobilized onto a CM5 chip (#BR-1005-30, GE) using anamine coupling kit (#BR-1000-50, GE) according to the manufacturer'sinstructions. After the immobilization, c-Met-Fc (#358-MT/CF, R&DSystems) was injected thereto at the amount of 15 μg/m for 1 min. Then,the bispecific antibody ME22S prepared in Example 2 was injected at theamount of 20 nM for 1 min. Thereafter, EGFR-Fc (#344-ER, R&D Systems)was injected at 100 nM for 1 min and then, association/dissociation wasobserved.

In addition, the affinities of the prepared anti-c-Met/anti-EGFRbispecific antibody to the two types of antigens, c-Met and EGFR, wereeach identified using Biacore T100 (GE). A human Fab binder (GEHealthcare) was immobilized at the surface of a CM5 chip (#BR-1005-30,GE) according to the manufacturer's specifications. About 90 to 120 Ruof ME22S was captured, and c-Met-Fc (#358-MT/CF, R&D Systems) or EGFR-Fc(#344-ER, R&D Systems) were injected at various concentrations into thecaptured antibody. 10 mM Glycine-HCl (pH 1.5) solution was injectedthereto to regenerate the surface. In order to measure affinity, thedata obtained from this experiment was fitted using BIAevaluationsoftware (GE Healthcare, Biacore T100 evaluation software).

The obtained results are shown in Table 5 and FIG. 3, wherein it isconfirmed that ME22S binds to both of the two antigens, c-Met and EGFR.

TABLE 5 Antibody Antigen K_(D) (nM) k_(a) (1/Ms) k_(d) (1/s) ME22S cMet0.05 1.3 × 10⁵ 5.5 × 10⁻⁵ EGFR 0.49 1.9 × 10⁵ 9.2 × 10⁻⁵

Example 5 Examination of Cancer Cell Proliferation Inhibition byAnti-c-Met/Anti-EGFR Bispecific Antibody

The cancer cell proliferation inhibition effects of theanti-c-Met/anti-EGFR bispecific antibody prepared in Example 2 wereexamined in stomach cancer cell lines SNU5 and SNU638, human skin cancercell line A431, human lung cancer cell line NCI-H1993, and human headand neck cancer cell lines HN3 and K2D. SNU5, SNU638, A431, NCI-H1993,and HN3 cell lines were all purchased from ATCC. K2D cell line, which isa patient derived cell line, was donated from Medical College of KonkukUniversity.

A1 cells except A431 were incubated in RPMI1640 medium (#11875-093,Gibco) supplemented with 10% (v/v) FBS and 1% (v/v)Penicillin-Streptomycin under the conditions of 5% CO₂ and 37° C. A431cells were incubated in DMEM medium (#30-2002, ATCC) supplemented with10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin under the conditionsof 5% CO₂ and 37° C. For cell proliferation assay, each cell line wassub-cultured at a concentration of 5×10³ cell/well in a 96-well plate,which was treated with the anti-c-Met/anti-EGFR bispecific antibodyME22S prepared in Example 2 in an amount of 0.25 ug (microgram)/ml (SNU5cell line) or 5 μg/m (SNU638, A431, and NCI-H1993 cell lines), andcultured for 72 hours. A medium with no antibody was used as a negativecontrol, and commercially available EGFR inhibitor Erbitux®(#ET509081213, Merck) treated group, L3-1Y-IgG2 antibody (prepared inreference example) treated group, and co-treated group of L3-1Y-IgG2 andErbitux® were each used as positive controls. The amount of Erbitux® andL3-1Y-IgG2 were 0.25 μg/ml for SNU5 and 5 μg/m for SNU638, A431, andNCI-H1993 cell lines. HN3 and K2D cell lines were treated with ME22S byserially diluting from 10 μg/ml to 5-fold dilution concentration andincubated for 72 hours.

After incubation, cell proliferation degrees were analyzed using CellCounting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, Md.)according to the manufacturer's instructions. In brief, after theincubation for 72 hours, 10 μl (microliter) of CCK8 solution was addedto each well and after the additional incubation for 2.5 hours,absorption degrees were read at 450 nm using a microplate reader.

The obtained results are shown in FIGS. 4A to 4F. As seen in FIGS. 4A to4F, the anti-c-Met/anti-EGFR bispecific antibody ME22S showed remarkableincreases in cell proliferation inhibitory effects, compared to thecases treated individually with the anti-c-Met antibody L3-1Y-IgG2 andthe anti-EGFR antibody Erbitux®. In particular, the anti-c-Met/anti-EGFRbispecific antibody ME22S showed excellent cell proliferation inhibitoryeffects, even compared to the co-treatment case (L3-1Y-IgG2+Erbitux®),which is not a form of bispecific antibody.

Example 6 Examination of Cancer Cell Proliferation Inhibition byAnti-c-Met/Anti-EGFR Bispecific Antibody in Resistance-Induced CancerCells

Cell lines N87(ATCC) and NCI-H820(ATCC) were incubated in RPMI1640medium supplemented with 10% (v/v) FBS and 1% (v/v)Penicillin-Streptomycin under the condition of 5% CO₂ and 37° C. Forcell proliferation assay, N87 cell line was sub-cultured at aconcentration of 5×10³ cell/well in a 96-well plate, which was treatedwith 500 nM of Lapatinib (sc-353686, Santa Cruz Biotechnology) alone,500 nM of Lapatinib and 1 μg/m of L3-1Y-IgG2 (co-treatment), 500 nM ofLapatinib and 1 μg/m of Erbitux® (co-treatment), and 500 nM of Lapatiniband 1 μg/m of ME-22S (co-treatment), and cultured for 72 hours. Forresistance induction, 100 ng/ml of HGF (PGR 101-200, Pangen) was addedto each of the above treatment groups and cultured for 72 hourstogether.

NCI-H820 cell line was sub-cultured at a concentration of 5×10³cell/well in a 96-well plate, which was treated with 5 μg/m of ME-22S,and cultured for 72 hours. A medium with no antibody was used as anegative control, and commercially available EGFR inhibitor Erbitux®(#ET509081213, Merck) treated group, L3-1Y-IgG2 antibody (prepared inreference example) treated group, and co-treated group of L3-1Y-IgG2 andErbitux® were each used as positive controls. The amount of each ofErbitux® and L3-1Y-IgG2 was 5 μg/m for each group. For resistanceinduction, 100 ng/ml of HGF (PGR 101-200, Pangen) was added to each ofthe above treatment groups and cultured for 72 hours together.

After incubation, cell proliferation degrees were analyzed using CellCounting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, Md.)according to the manufacturer's instructions. In brief, after theincubation for 72 hours, 10 μl of CCK8 solution was added to each welland after the additional incubation for 2.5 hours, absorption degreeswere read at 450 nm using a microplate reader.

The obtained results are shown in FIG. 5. As seen in FIG. 5, theanti-c-Met/anti-EGFR bispecific antibody ME22S showed remarkableincreases in cell proliferation inhibitory effects on N87 and H820 celllines where a resistance is induced by HGF treatment as well as N87 andH820 cell lines treated with no HGF.

Example 7 C-Met and EGFR Activation Inhibitory Effect ofAnti-c-Met/Anti-EGFR Bispecific Antibody

SNU638 cells were treated with Erbitux®, L3-1Y-IgG2 (ReferenceExample 1) and ME22S (Example 2), alone or in combination, for 40minutes. Then each of HGF and EGF (236-EG-200, R&D systems) was addedthereto at the amount of 100 ng/ml. Then, the cells were lysed withComplete Lysis-M (#04719956001, Roche), and the resulted cell lysateswere collected and subject to SDS-PAGE electrophoresis.

After the electrophoresis, the proteins from the gels were transferredonto a nitrocellulose membrane (#LC2006, Invitrogen), which was thenblocked with a skim milk (5% in TBST) at a room temperature for onehour. After blocking, for the primary antibody reaction, the membranewas treated with a 1:1000 dilution of anti-phospho c-Met antibody,anti-phospho EGFR antibody, anti-EGFR antibody, anti-phosphor Aktantibody, anti-Akt antibody (all available from Cell signalingtechnology), and anti-c-Met antibody (Abcam) in an amount of 10 μg/ml ata room temperature for two hours. After the primary antibody reaction,the membrane was washed three times with PBS for 5 min. and then, it wastreated with a horseradish peroxidase attached secondary antibody (Cellsignaling technology) against each primary antibody in an amount of 1μg/ml at a room temperature for one hour. After the secondary antibodyreaction, the membrane was washed three times with PBS for 5 min. andthen sensitized with Enhanced chemiluminescence substrate (Thermoscientific) to measure the degree of antigen-antibody reaction byImageQuant LAS system (GE Healthcare).

The obtained results are shown in FIG. 6. As shown in FIG. 6, thetreatment of the anti-c-Met/anti-EGFR bispecific antibody ME22S resultsin increased c-Met phosphorylation inhibition and c-Met degradation,compared with treatment of anti-c-Met antibody L3-1Y-IgG2 alone, andL3-1Y-IgG2 and EGFR inhibitor Erbitux® in combination. In addition, whenEGFR inhibitor Erbitux® is treated alone or in combination withL3-1Y-IgG2, EGFR phosphorylation inhibition is observed, but EGFRdegradation is nearly not observed. When the anti-c-Met/anti-EGFRbispecific antibody ME22S is treated, EGFR degradation as well as EGFRphosphorylation inhibition is observed. Such results indicate that theanti-c-Met/anti-EGFR bispecific antibody ME22S has EGFR degradationactivity as well as EGFR phosphorylation inhibitory activity, indicatingthat an EGFR inhibitor such as anti-EGFR antibody can possess asynergistic effect by forming a bispecific antibody with an anti-c-Metantibody, compared to an EGFR inhibitor alone.

Example 8 Co-Localization of c-Met and EGFR by Anti-c-Met/Anti-EGFRBispecific Antibody

SNU638 cells (ATCC) were provided at the amount of 4×10⁴ cell/well, andtreated with L3-1Y-IgG2 and anti-c-Met/anti-EGFR bispecific antibodyME22S alone or in combination at the amount of 5 μg/m per well (in thecase of combination treatment, the amount of each antibody is 5 μg/m)under 37° C. for 4 hours. The cells were treated with 4% (v/v)formaldehyde for 15 minutes to be immobilized on a plate, and washedthree times with PBS. Thereafter, the cells were treated with a blockingbuffer (0.5% triton x-100 and 5% donkey serum) at a room temperature forone hour and then treated with a 1:100 dilution of primary antibodiesagainst each of c-Met and EGFR (c-Met primary antibody; #FAB3582A, R&Dsystems, EGFR primary antibody; #5616, Cell signaling) against c-Met andEGFR, respectively in the amount of 100 μl, at 4° C. for 15 hours. Afterthe cells were washed three time with PBS, they were treated with a1:2000 dilution of a secondary antibody (#A21433, Invitrogen) in anamount of 100 ul at a room temperature for 1 hour and washed three timeswith PBS to prepare a plate with a mounting medium (#H-1200, Vector).The prepared cells were observed with a confocal microscope (Zeiss,LSM710).

The obtained results are shown in FIG. 7. As seen in FIG. 7, when EGFRinhibitor Erbitux® is treated alone, EGFR locates on cell membranes, andeven when L3-1Y-IgG2 and Erbitux® are treated together, c-Met migratesto inside of the cell but EGFR still locates on cell membrane. However,when the anti-c-Met/anti-EGFR bispecific antibody ME22S is treated, bothof c-Met and EGFR migrate to inside of the cells.

Example 9 Decrease in Expression of Target Receptor byAnti-c-Met/Anti-EGFR Bispecific Antibody

To examine decrease of expressed level of target receptors, c-Met andEGFR by ME22S, human stomach cancer cell line, MKN45 (ATCC) and SNU 638(ATCC) were sub-cultured on 96-well plate, where the concentration ofeach cell line is 2×10³ cell/well. The cells were treated with each ofL3-1Y-IgG2, Erbitux®, and ME22S, at the amount of 5 μg/ml, and culturedfor 24 hours. A medium with no antibody was used as a negative control.After culture, the cells were lysed with Complete Lysis-M (#04719956001,Roche), and cell lysates were collected. Total cMet detection ELISA kit(DYC358E, R&D systems) was used for measuring the expression level ofc-Met, and Total EGF Receptor ELISA kit (#7297, Cell Signaling) formeasuring the expression level of EGFR, according to manufacturer'sindications.

The obtained results are shown in FIG. 8. As shown in FIG. 8, L3-1Y-IgG2and ME22S considerably decrease the expression level of c-Met. Erbitux®does not effect on the expression level of EGFR, whereas ME22Sconsiderably decreases the expression level of EGFR as well as theexpression level of c-Met.

Example 10 Inhibition of Cancer Metastasis by Anti-c-Met/Anti-EGFRBispecific Antibody

Human stomach cancer cell line, SNU638 cells (ATCC) were sub-cultured ontrans-well CIM-16 plate (Roche Applied Bioscience) in the concentrationof 1×10⁵ cell/well. During the sub-culture, L3-1Y-IgG2, Erbitux®, andME22S were treated alone or in combination, at the amount of 10 μg/ml,and to induce metastasis, HGF and EGF were treated at the amount of 100ng/ml, respectively, simultaneously with the antibody treatment. Theratio of cancer metastasis was measured in real time using xCelligenceRTCA DP instrument (Roche Applied Biosience).

The obtained results are shown in FIG. 9. As shown in FIG. 9, thetreatment of Erbitux® alone has no effect in inhibiting cancermetastasis, but the treatment L3-1Y-IgG2 alone and the combinationtreatment of L3-1Y-IgG2 and Erbitux® result in inhibiting cancermetastasis to the ratio of about 30%. However, ME22S exhibits moreexcellent inhibition ratio of cancer metastasis as about 45%, indicatingthat the anti-c-Met/anti-EGFR bispecific antibody is more effective ininhibition of cancer metastasis.

Example 11 Cancer Cell Inhibition Effect of Anti-c-Met/Anti-EGFRBispecific Antibody

SNU5 cells (ATCC) were injected into dorsal subcutis of 5-6 week oldCB.17-SCID mice (Shanghai SLAC Laboratory Animal Co. Ltd.) at the amountof 3×10⁶ cell/mouse, to prepare tumor models. When the tumor volumereaches 100 mm³, 1 mg/kg of L3-1Y-IgG2 and 1 mg/kg ofanti-c-Met/anti-EGFR bispecific antibody ME22S were intravenouslyadministered alone or in combination once a week for 4 weeks in total. Agroup where PBS is intravenously administered thereto was used as anegative control, and a group where 10 mg/kg of an anticancer drug,Irinotecan (Pfizer Inc.), was intravenously administered thereto twice aweek for 4 weeks in total, was used as a positive control. The tumorsize (length×breadth×height) was measured twice each week, and at thelast week, the mice were autopsied.

The obtained tumor size is shown in FIG. 10. As shown in FIG. 10, whenL3-1Y-IgG2 is treated alone, the tumor growth is inhibited and the finaltumor size is nearly not increased from the initial size of 100 mm³, andthe positive control shows the similar results where the final tumorsize is about 72 mm³. However, when ME22S is treated, the tumor sizebecomes significantly decreased from 7 days after the administration,and the final tumor size is about 12 mm³. These results indicate thatthe anti-c-Met/anti-EGFR bispecific antibody has more increasedinhibitory effect on cancer cell growth compared to L3-1Y-IgG2 andpre-existing drug, Irinotecan.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. An anti-EGFR antibody or an antigen-bindingfragment thereof, comprising: a CDR-H1 comprising the amino acidsequence of SEQ ID NO: 109, a CDR-H2 comprising the amino acid sequenceof SEQ ID NO: 110, a CDR-H3 comprising the amino acid sequence of SEQ IDNO: 111; a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 112,a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 113, and aCDR-L3 comprising the amino acid sequence of SEQ ID NO:
 114. 2. Theanti-EGFR antibody or an antigen-binding fragment thereof according toclaim 1, comprising: a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 115 or SEQ ID NO: 117, a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 116 orSEQ ID NO: 118, or a combination thereof.
 3. The anti-EGFR antibody oran antigen-binding fragment thereof according to claim 2, wherein theanti-EGFR antibody or an antigen-binding fragment is an anti-EGFR scFvcomprising: a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 115 or SEQ ID NO: 117, and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 116 or SEQ IDNO:
 118. 4. An anti-c-Met/anti-EGFR bispecific antibody comprising ananti-c-Met antibody or an antigen-binding fragment thereof and ananti-EGFR antibody or an antigen-binding fragment thereof, wherein theanti-c-Met antibody or an antigen-binding fragment thereof comprises aCDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H2comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2, aCDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO:3; a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15; andthe anti-EGFR antibody or an antigen-binding fragment thereof comprises:a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 109, a CDR-H2comprising the amino acid sequence of SEQ ID NO: 110, a CDR-H3comprising the amino acid sequence of SEQ ID NO: 111; a CDR-L1comprising the amino acid sequence of SEQ ID NO: 112, a CDR-L2comprising the amino acid sequence of SEQ ID NO: 113, and a CDR-L3comprising the amino acid sequence of SEQ ID NO:
 114. 5. Theanti-c-Met/anti-EGFR bispecific antibody according to claim 4, whereinthe antigen-binding fragment of the anti-c-Met antibody or the anti-EGFRantibody is selected from the group consisting of scFv, (scFv)2, scFvFc,Fab, Fab′, and F(ab′)2.
 6. The anti-c-Met/anti-EGFR bispecific antibodyaccording to claim 4, wherein the anti-c-Met antibody comprises: aCDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:22, SEQ ID NO: 23, or SEQ ID NO: 24, a CDR-H2 comprising the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 25, or SEQ ID NO: 26, a CDR-H3comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 27, SEQID NO: 28, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, or SEQ ID NO: 106, a CDR-L2 comprising the amino acid sequence ofSEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, and aCDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
 37. 7. Theanti-c-Met/anti-EGFR bispecific antibody according to claim 4, whereinthe anti-c-Met antibody or the antigen-binding fragment thereofcomprises: a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94, a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 18, 19,20, 21, 75, 88, 95, 96, 97, 98, 99, 107, or 121, or a combination of theheavy chain variable region and the light chain variable region.
 8. Theanti-c-Met/anti-EGFR bispecific antibody according to claim 4, whereinthe anti-c-Met antibody comprises: a heavy chain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 62, theamino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO:62, SEQ ID NO: 64, the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64, SEQ ID NO: 66, and the amino acid sequencefrom the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and a lightchain comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 68, the amino acid sequence from the 20 to240^(th) positions of SEQ ID NO: 68, SEQ ID NO: 70, the amino acidsequence from the 20 to 240^(th) positions of SEQ ID NO: 70, and SEQ IDNO:
 108. 9. The anti-c-Met/anti-EGFR bispecific antibody according toclaim 4, wherein the anti-EGFR antibody or the antigen-binding fragmentthereof comprises: a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 115 or SEQ ID NO: 117, a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 116 orSEQ ID NO: 118, or a combination thereof.
 10. The anti-c-Met/anti-EGFRbispecific antibody according to claim 4, wherein the anti-EGFR antibodyor the antigen-binding fragment thereof is linked to C-terminus of theanti-c-Met antibody.
 11. A pharmaceutical composition comprising theanti-EGFR antibody or the antigen-binding fragment thereof of claim 1and a pharmaceutically acceptable carrier.
 12. A pharmaceuticalcomposition comprising the anti-c-Met/anti-EGFR bispecific antibody ofclaim 4 and a pharmaceutically acceptable carrier.
 13. Theanti-c-Met/anti-EGFR bispecific antibody according to claim 4, whereinthe anti-c-Met antibody or the antigen-binding fragment thereofcomprises: a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, 107, or 121; and theanti-EGFR antibody or the antigen-binding fragment thereof comprises: aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 115 or SEQ ID NO: 117, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 118.